Colony stimulating factors (CSFs) stimulate the differentiation and/or proliferation of bone marrow cells. CSFs in both human and murine systems have been identified and distinguished according to their activities involving two of the three main classes of leukocytes, namely granulocytes and monocytes. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively, while granulocyte-macrophage CSF (GM-CSF) has broader activities and stimulates the formation of both macrophage, neutrophilic, and eosinophilic granulocyte colonies. These CSFs act via their respective receptors, namely G-CSFR, M-CSFR, and GM-CSFR. G-CSR is expressed on multipotential hematopoietic progenitor cells and cells of myeloid lineage, and is important for regulation of granulopoiesis.
Evidence of the role G-CSF and G-CSFR play in inflammation includes the discovery that G-CSF is frequently found elevated in serum of and at inflammatory sites in patients with infections. The undetectable normal circulating levels of G-CSF (≦10 pM) increase in inflammatory conditions to a range of from 100 to 2000 pM. Further, transgenic mice with neutrophils expressing chimeric receptors with extra-cellular G-CSFR and intra-cellular erythropoietin receptor appear to retain their normal hematopoietic function but no longer respond to chemotactic signals. Also, the chemokine interleukin-8 (IL-8) fails to induce chemotaxis of neutrophils from G-CSFR −/− mice (i.e., G-CSFR knockout mice), suggesting a specific role for G-CSFR in neutrophil chemotaxis. However, by itself, G-CSF is a relatively weak chemoattractant.
Additionally, M-CSF, also known as colony stimulating factor-1, has been shown to increase blood and tissue macrophage numbers in several species. For example, it is known that M-CSF is produced within the joint in human rheumatoid arthritis, where it has been shown to cause severe exacerbation of the disease. This is consistent with other studies, wherein M-CSF was found to worsen the disease course of experimental disseminated candidiasis, a disease with many of the characteristics of tumor necrosis factor-mediated pathology. M-CSF was also found to stimulate secretion of urokinase plasminogen activator, which plays a role in proteolytic joint destruction. Recently, cDNA encoding the primary growth and differentiation factor for M-CSF has been isolated, sequenced and expressed, and human recombinant M-CSF is now available for experimental studies.
However, CSFs are not the only cytokines involved in inflammation. Also involved are chemokines, which are chemotactic cytokines that are released by a wide variety of cells to attract macrophages, T cells, eosinophils, basophils, and neutrophils to sites of inflammation. There are two classes of chemokines, the members of each class share an organizing primary sequence motif. Alpha chemokines such as IL-8, neutrophil-activating protein-2 (NAP-2), and melanoma growth stimulatory activity protein (MGSA) are chemotactic primarily for neutrophils, whereas beta chemokines such as RANTES (regulation-upon-activation, normal T expressed and secreted), MIP-1 alpha (macrophage inflammatory protein), MIP-1 beta, MCP-1 (monocyte chemotactic protein-1), MCP-2, and MCP-3 are chemotactic for monocytes, T-cells, eosinophils, and basophils.
Chemokines bind specific cell-surface receptors belonging to the family of G-protein-coupled seven-transmembrane-domain proteins which are termed “chemokine receptors.” Chemokines and chemokine receptors such as, for example, CCR-1, CCR-2, CCR-2a, CCF-2b, CCR-3, CCR-4, CCR-5, CXCR-1, CXCR-2, CXCR-3, and CXCR-4, play a role in inflammation and autoimmune responses by attracting leukocytes, which migrate out of the microvasculature and into the extravascular space in response to chemoattractant molecules. These chemoattractants, which include cytokines and activated complement components, may be released by the patient or they may be released from an invading organism. Once exposed to chemoattractants within the vasculature, the leukocytes become activated and capable of adhering to the endothelium, providing the first step in the development of inflammation. Stimulated neutrophils adhere to the endothelium of the microvasculature in response to a gradient of chemoattractants which direct the cells into the extravascular space toward the source of the chemoattractant.
One chemokine in particular that mediates inflammatory response is IL-8. IL-8 is a cytokine that promotes the recruitment and activation of neutrophil leukocytes and represents one of several endogenous mediators of the acute inflammatory response. In the past it was variously termed neutrophil-activating factor, monocyte-derived neutrophil chemotactic factor, IL-8, and neutrophil-activating peptide-1. The term “IL-8” has gained the widest acceptance and will be used herein.
Evidence of the involvement of IL-8 in inflammatory responses includes the observation that neutralizing antibodies to human IL-8 were shown to have a protective effect in inflammatory lung injury in rats.
Further, preliminary nonhuman primate studies have confirmed the activity of IL-8 on hematological parameters. IL-8 was administered by both bolus and continuous infusion to baboons. This resulted in a rapid, transient and severe granulocytopenia followed by granulocytosis that persisted as IL-8 levels remained detectable within the circulation. Histopathological examination revealed a mild to moderate neutrophil margination in the lung, liver and spleen which was of greater severity in animals receiving the continuous infusion of IL-8.
Also, high levels of intravascular IL-8 have been reported in systemic conditions such as septic shock.
Further, it is known that IL-8 binds with a higher affinity to CXCR-1 than to CXCR-2. On the other hand, a primary receptor for MCP-1 is CCR-2, which is expressed predominately on macrophages.
Another chemokine that mediates inflammatopry response is MCP-1. Studies using animal macrophages have demonstrated the pivotal roles of MCP-1 in rheumatoid arthritis and atherosclerosis. Unfortunately, work on human macrophages has been hampered by the relative small number of these cells in human blood.
Historically, persons skilled in the pharmaceutical and medical arts have sought to increase levels of CSFs in patients, believing that CSFs provided therapeutic benefits to patients suffering from certain diseases and disorders. We have now unexpectedly discovered that CSFs synergistically enhance the chemoattractant effects of chemokines on recruitment of leukocytes to sites of inflammation. For example, it is shown below that G-CSF synergistically enhances the chemoattractant effects of IL-8 on the recruitment of neutrophils, and M-CSF synergistically enhances the chemoattractant effects of MCP-1 on the recruitment of monocytes. As IL-8 and MCP-1 are key mediators of inflammatory diseases, it would be desirable to identify substances capable of inhibiting the synergistic interactions of CSFs and chemokines for use in the treatment of diseases responsive to this inhibition.
We have now unexpectedly discovered useful methods for determining the ability of a compound, or a pharmaceutically acceptable salt thereof, to inhibit a synergistic interaction between a CSF and a chemokine, including a method for rapidly screening large numbers of such compounds. Accordingly, one embodiment of the present invention is a method for screening compounds, or pharmaceutically acceptable salts thereof, for inhibition of a synergistic interaction between a CSF and a chemokine. All that is needed to practice this embodiment of the present invention is to assay a potential inhibitor of said synergistic interaction according to the methods described below.
Another embodiment of the present invention is an inhibitor of a synergistic interaction between a CSF and a chemokine, which inhibitor is identified using a screening method of the present invention.
Further, another embodiment of the present invention is an inhibitor of a synergistic interaction between a CSF and a chemokine.
Still further, another embodiment of the present invention is a method of treating diseases and disorders responsive to inhibition of a synergistic interaction between a CSF and a chemokine.
Still further, another embodiment of the present invention is a pharmaceutical composition comprising an inhibitor of a synergistic interaction between a CSF and a chemokine, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
All that is needed to practice the present invention is to administer from one to six times daily a therapeutically effective amount of an inhibitor, or a pharmaceutically acceptable salt thereof, of a synergistic interaction between a CSF and a chemokine to a patient in need thereof for the treatment of inflammatory disorders and diseases responsive to inhibition of a synergistic interaction between a CSF and a chemokine. Determination of proper dosage, pharmaceutically composition, and form of administration of the inhibitor is well within ordinary skill in the pharmaceutical and medical arts.
U.S. Pat. No. 4,504,586 discloses murine-derived hybridoma tumor cell lines and monoclonal anti-Colony Stimulating Factor Subclass Number 1 antibody substances produced by these cell lines. Use of said monoclonal antibody substances, alone or in combination, in immunological procedures for isolation of natural Colony Stimulating Factor Subclass Number 1 and for quantitative detection of colony Stimulating Factor Subclass Number 1 in fluid samples.